The hepatotoxicity of acetaminophen is believed to be mediated by the reactive metabolite, N-acetylimidoguinone. At therapeutic doses of acetaminophen, the reactive metabolite is efficiently detoxified by reaction with glutathione (GSH). An acetaminophen-GSH conjugate is believed to be formed but definitve proof of its structure has not yet been presented until now. Acetaminophen was administered to rats and a metabolite believed to be acetaminophen-GSH conjugate was isolated from bile by preparative high pressure chromatography. The 13C NMR and field desorption mass spectometry of the metabolite confirmed that it was a GSH conjugate of acetaminophen. A 1H NMR investigation of the metabolite established uniquivocally that the GSH-conjugate of acetaminophen is 3-(glutathion-S-yl) acetaminophen. This finding is consistent with N-acetylimidoguinone being the reactive metabolite of acetaminophen that reacts with liver GSH, and other tissue molucules.